Unique Domain for a Unique Target: Selective Inhibitors of Host Cell DDX3X to Fight Emerging Viruses

Article abstract of DOI:10.1021/acs.jmedchem.0c01039

Emerging viruses like dengue, West Nile, chikungunya, and Zika can cause widespread viral epidemics. Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses exploit common cellular

Full text of DOI:10.1021/acs.jmedchem.0c01039

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Article
A unique domain for a unique target: selective
inhibitors of host cell DDX3X to fight emerging viruses.
Valentina Riva, Anna Garbelli, Annalaura Brai, Federica Casiraghi, Roberta Fazi,
Claudia Trivisani, Adele Boccuto, Francesco Saladini, Ilaria Vicenti, Francesco Martelli,
Maurizio Zazzi, Simone Giannecchini, Elena Dreassi, Maurizio Botta, and Giovanni Maga
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01039 • Publication Date (Web): 30 Jul 2020
Downloaded from pubs.acs.org on August 3, 2020
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Journal of Medicinal Chemistry
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A unique domain for a unique target: selective inhibitors of host cell
DDX3X to fight emerging viruses.
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Valentina Riva1‡, Anna Garbelli1‡, Annalaura Brai2‡, Federica Casiraghi1†, Roberta Fazi2, Claudia
I. Trivisani2, Adele Boccuto3, Francesco Saladini3, Ilaria Vicenti3, Francesco Martelli4, Maurizio
Zazzi3, Simone Giannecchini4, Elena Dreassi2, Maurizio Botta2,5¶ and Giovanni Maga1*
1
Istituto di Genetica Molecolare IGM-CNR "Luigi Luca Cavalli-Sforza", Via Abbiategrasso 207, I-27100 Pavia, Italy.
2
Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy.
3
Dipartimento di Biotecnologie Mediche, Università degli Studi di Siena, Viale Bracci 16, I-53100 Siena, Italy.
4
Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi di Firenze, Viale Morgnani 48, I-50134 Firenze,
Italy.
5
Biotechnology College of Science and Technology, Temple University, Biolife Science Building, Suite 333, 1900 N 12th
Street, Philadelphia, Pennsylvania 19122.
KEYWORDS: Antiviral inhibitors/DDX3X/Emerging viruses/RNA helicase/Unique domain
ABSTRACT: Emerging viruses like Dengue, West Nile, Chikungunya and Zika can cause widespread viral epidemics.
Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses
exploit common cellular pathways opening the possibility to develop broad-spectrum antiviral agents targeting host factors.
The human DEAD-box RNA helicase DDX3X is an essential cofactor for viral replication but dispensable for cell viability.
Herein, we exploited the presence of a unique structural motif of DDX3X not shared by other cellular enzymes to develop a
theoretical model to aid in the design of a novel class of highly selective inhibitors acting against such specific target, thus
limiting off-targeting effects. High throughput virtual screening led us to identify hit compound 5, endowed of promising anti-
enzymatic activity. To improve its aqueous solubility, 5 and its two enantiomers were synthesized and converted into their
corresponding acetate salts (compounds 11, 12 and 13). In vitro mutagenesis and biochemical and cellular assays further
confirmed that the developed molecules were selective for DDX3X and were able to suppress replication of West Nile and
Dengue viruses in infected cells in micromolar range while showing no toxicity for uninfected cells. These results provide
proof-of-principle for a novel strategy in developing highly selective and broad-spectrum antiviral molecules active against
emerging and dangerous viral pathogens. This study paves the way for the development of larger focused libraries targeting
such domain, in order to expand SAR studies and fully characterize their mode of interaction.
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However, to further develop such compounds for human use, it
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Introduction
is of paramount importance to endow them with chemical and
structural features able to guarantee an absolute selectivity for
DDX3X only. This has proven a challenging task, due to the
significant similarity both from a sequence and a structural
point of view, in the ATP- and RNA-binding sites among the
Despite much social visibility and alertness to the impact of
widespread viral epidemics, a significant number of emerging
viral pathogens remain without effective treatment or cure. For
example, no specific and effective pharmacological treatments
are currently available for diseases caused by Dengue virus
1
6
members of the DEAD-box family of RNA helicases . The name
of the family derives from the Walker B motif II D-E-A-D (Asp–
Glu–Ala–Asp), which contains the catalytic residues essential
for ATP hydrolysis. In all DEAD-box helicases characterized to
date, the ATPase and RNA helicase motifs involved in RNA
substrate binding and hydrolysis are distributed in two RecA-
like subdomains. Also in DDX3X, all the 12 canonical motifs
common to all members of the DEAD-box RNA helicase family
are present in both domains (Domain 1 and Domain 2, from N-
(
(
DENV), West-Nile virus (WNV), Japanese encephalitis virus
JEV), Chikungunya virus (CHIKV) and Zika virus (ZIKV), while
it is becoming clear that they pose an increasing global health
threat and could eventually lead to sustained epidemics
worldwide .
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The current view of the infected cell as a "virocell" , reflects the
notion that the metabolism of the infected cells is completely
remodelled and redirected to produce new viral particles. The
extent of such reprogramming has been well illustrated by
transcriptome- and proteome-level studies in cells infected by
different viruses, showing that the limited number of viral
proteins transcribed from the viral mRNAs, can interact with
hundreds of cellular proteins, often located at the nodes of
11
to C-terminal) connected via a short flexible linker . DEAD-box
helicases, including DDX3X, also contain additional specific
motifs. The Q-motif establishes specific contacts with the
adenine base of ATP, while two additional motifs, Ib (also
termed GG-motif) and IVa (or QxxR-motif), are involved in
3
14, 17
complex metabolic networks . A key observation stemming
specific contacts with the RNA substrate
. In DDX3X, an
from these studies is that many different viruses rely on the
same cellular proteins for modulating the host cell response.
One of these proteins is the human X-linked DEAD-box protein
additional ATP-interacting domain (ABL, aa 135-168) is
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involved in RNA stimulation of ATP hydrolysis .
In addition to all the canonical conserved domains, DDX3X has
been shown to possess a unique insertion hence named
“unique motif” (sequence -ALRAMKENGRYGRRK- aa 250-264),
between motif I (Walker A), which is involved in ATP binding,
and motif Ia, which is part of the RNA binding domain.
Unfortunately, all the available crystal structures of DDX3X do
not allow to have detailed information around this unique
3
(DDX3X).
DDX3X is involved in different cellular pathways (translation,
4,
5
transcription, RNA decay, ribosome biogenesis)
. It
participates in cell cycle control, apoptosis, oncogenic
transformation, cell migration and hypoxia, and is
overexpressed in a large number of cancers and has been
proposed as a novel target for the development of anticancer
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motif. In the structure of Schütz and collaborators DDX3X
crystal lacks bound RNA, thus representing the "open"
conformation of the enzyme and making it difficult to
determine the precise nature of the contacts between the
unique motif (UM) residues and the nucleic acid lattice. Vice
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-8
therapeutics . DDX3X inhibitors have been identified with
specific anticancer activities in mouse models, without
9,10
showing long-term toxic effects to the animals . Given the
high homology between human and murine DDX3X (98.6%),
these results strongly support the notion that inhibiting DDX3X
should not result in major adverse effects in humans also,
providing proof of principle that DDX3X inhibitors can be used
to selectively target cancer with minimal toxicity for normal
cells.
DDX3X is also an essential host factor for the replication of
viruses belonging to different families, such as Herpesviridae,
Retroviridae, Flaviviridae, Poxviridae, Caliciviridae, where it
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versa, in the more recent structure of Song and Ji active
conformation, the UM region is not present probably because
its very mobile nature which make the crystallization a difficult
task.
Despite the scarce structural information, such insertion
represents a very attractive target, since it is not present in
21
other human members of the DEAD-box family and BLAST
analysis indicated that no other human protein shares
significant homology with the aminoacidic sequence of the UM.
We have previously shown that deleting this short motif
affected the RNA helicase activity of DDX3X, by reducing its
affinity for the nucleic acid and that blocking this domain with
1
1, 12
plays different roles in their life cycle
. DDX3X participates
also in the host innate immunity. It has been shown to interact
with and regulate the protein kinases IKKɛ and TBK-1, involved
in IFN-β production, as well as to regulate the NF-κB dependent
inflammatory response . However, how DDX3X activity is
coordinated in the different branches of the innate immune
response and to what extent its role is essential in the global
antiviral response of the cells is still not completely
understood. In recent years, different groups, including ours,
have extensively studied DDX3X as a target to control viral
infections and developed small molecule inhibitors targeting
both the ATP- and RNA-binding sites of DDX3X . In particular,
we have identified a RNA-competitive inhibitor targeting the
nucleic acid binding cleft of DDX3X, which was able to block the
replication of Human immunodeficiency virus type 1 (HIV-1)
as well as Hepatitis C virus (HCV), DENV, WNV; and this despite
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a peptide reduced HIV-1 replication in infected cells . These
results suggested that inhibitors targeting the UM could be
highly selective for DDX3X, due to the absence of such domain
in other human proteins and might retain the ability to
suppress viral replication.
In light of these preliminary data, we decided to make the UM
the focus of a novel strategy to develop highly selective and
broad-spectrum antivirals, by designing small molecules
specifically targeting this unique DDX3X motif.
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Results
Molecular modeling
Structure-guided drug design is a powerful approach to
develop molecules specifically tailored to a target. However,
the reliability of such an approach relies on the availability of
high-resolution structures of the desired target. Unfortunately,
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the potential antiviral role of DDX3X in innate immunity .
Preliminary in vivo studies in rats, showed no toxic effects for
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this compound . This suggests that targeting DDX3X could be
a powerful method to halt the replication of different viruses.
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at the time of the starting of this project, no structures of
were first docked within our selected pocket using GLIDE with
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DDX3X in its active conformation were available. Thus, in order
to overcome such limitation and to study the human DDX3X
active closed conformation, we built a homology model using
as first template the crystal structure of Drosophila Vasa DEAD-
box helicase (PDB code: 2DB3) which is co-crystallized with
AMPPNP and an RNA fragment and shows a 44% sequence
homology with DDX3X. Since the DDX3X unique motif is
missing in the 2DB3 structure, the crystal structure of DDX3X
in the open conformation (PDB code: 2I4I) was chosen as
second template to fill the missing region. The homology model
was built using the software PRIME.
the HTVS procedure, which allowed us to analyze a large
number of molecules. From this analysis, we chose the best
scoring molecules (74,783: 20% of total) to be processed in the
next step.
Selected molecules were docked again within the pocket using the
docking program GOLD (CHEMSCORE). The following
parameters were used to detect the box grid for the docking study:
cavity centered by atom Arg252; radius 9 Å. Docked molecules
were then selected based on: i) score values above 23; ii) the
number of clusters generated (less than 7), iii) visual inspection.
Because of the small and narrow properties of the pocket, a fair
number of compounds were docked outside and so they were
discarded. Among the molecules which passed the filter analysis
and were docked inside the pocket (about 103), 10 compounds were
finally selected as interesting, based on their chemical structures
and their polar interactions into the active site.
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The recent structure of Song and Ji of DDX3X in its active
conformation does not contain the UM region. So, we decided
to build the UM homology model (Figure S1) using the software
PRIME and as template the crystal structure of DDX3X in the
open conformation (PDB code: 2I4I). The RMSD calculated
between DDX3X domains containing the UM region in the
homology model and in the crystal structure is 1.03 Å,
indicating the reliability of the modelling approach. Moreover,
as reported by Song and Ji, our homology model represents the
post-unwound state of the DDX3X protein. Our approach has
obvious limitations, being based on homology modelling, even
though developed with the most accurate tools and
appropriate templates available. However, it represents the
only available theoretical framework to start exploring the
druggable properties of the UM subdomain of DDX3X.
Identification of hit compounds
These molecules were then tested for their ability to inhibit
DDX3X RNA helicase activity in enzymatic assays. Table 1 lists
the chosen compounds with their structures, docking scores
and anti-enzymatic activities.
Table 1. Inhibitory activity and docking score of
selected compounds targeting the UM of DDX3X
Docking
score
(CHEMSCORE)
Thus, starting from our homology model, using PocketPicker, a
pymol plugin able to detect all the possible pockets on a protein
surface, we found a potential little pocket lined by the α-helix
formed by the DDX3X unique motif (Figure 1A and 1B).
Cmpd
ID
ID50
(µM)
a
QP
Chemical structure
logS
N
-
1
2
3
O
N
n.a
25.22
24.76
25.15
3.519
H
NO2
N
-
n.a
n.a
O
3.852
HO
-
4
N
.276
Figure 1. Molecular modeling of the inhibitor binding
pocket. A. Pockets identified by PockerPicker are represented
in dots. The unique motif UM (cyan) with the adjacent pocket is
circled. B. Zoom on the little pocket around the unique motif
NH2
-
4
5
N
n.a
24.83
NH
2.896
N
(
cyan). C-D. Two different orientations of compound 5 docked
H
pose showing, respectively, its particular shape profile and its
specific polar interactions with residues within the motif.
Templates used were PDB codes 2DB3 and 2I4I.
N
OH
-
0.7±0.2 24.68
Since no molecules able to bind this pocket within the UM have
ever been described, we performed a preliminary screening to
search theoretically active scaffolds among two commercial
libraries (Asinex Gold and Platinum consisting of 583,040
molecules) using two different docking programs. Molecules
2.126
NH2
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amino acids in the pocket (Glu256, Tyr266, Cys317). Another
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group of compounds (1, 2, 7) was selected considering the
shape and the residues involved in the hydrophobic
interactions that are Arg262, His318 and Pro247.
O
O
N
S
-
6
7
n.a
n.a
25.82
27.27
NH2
3.355
Based on our theoretical model, compound 5 was hypothesized
to have an interesting shape that fits into the pocket like a 'cap'.
Remarkably, compound 5 is the only one among the selected
molecules characterized by the presence of a hydroxyl group
possibly able to interact with both Glu256 and Cys317 (Figure
1C and 1D). As shown in Figure 2, the visual analysis of the
binding mode of compounds 1 and 7, characterized by the high
score but inactive in the biochemical assay, showed that these
molecules are not able to interact with Cys317, Tyr266, Glu256.
These last interactions characterize the binding mode of
compounds 5, 11, and 13 and are probably responsible for
their activity. To confirm the activity of this commercially
available molecule, compound 5 was re-synthesized as
depicted in Scheme 1, and converted into the corresponding
acetate salt (compound 11, Table 1). This simple synthetic
O
-
5
O
N
N
H
.331
O
N
0
1
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0
O
O
S
-
8
200±30 24.94
2.424
H2N
H2N
modification
provides
a
water-soluble
derivative.
Furthermore, the R and S enantiomers (compounds 12 and 13,
Table 1), were synthesized as described in Scheme 1.
N
-
3
9
n.a
n.a
25.68
25.86
.437
.632
N
NH2
-
1
10
N
O
Figure 2. In magenta is reported the binding mode of
Compound 1. The compound makes a hydrogen bond with
Arg262 and is involved in a π-π stacking interaction with
His266. Compound 7 is reported in yellow and is involved in
hydrogen bonds with Arg262 and Glu249 and in π-π stacking
interactions with His266.
N
0.002 ±
0.0005
-
1
1
1
1
2
3
24.68
24.28
21.65
NH2 CH3COOH
2.126
OH
More in detail epoxides 15a-c were produced starting from
N
0.007 ±
0.0001
-
diphenylamine
that
was
alkylated
with
racemic
NH2 CH3COOH
2.126
epichlorohydrin or with (R)-(-) or (S)-(+) epichlorohydrin.
Propanolamines 5, 16a and 16b were then obtained by
reaction with gaseous ammonia and subsequent treatment
with acetic acid to furnish the corresponding water-soluble
acetate salts 11-13 (Scheme 1).
OH
N
0.006 ±
-
NH2 CH3COOH
0
.0001
2.126
OH
a. ID50, inhibitor concentration reducing by 50% the enzyme
activity. Values are the mean of three independent replicates
±SD.
We then used our theoretical homology model to make
hypotheses on the kind of structural features which might
account for their activity. A first group of these molecules (4, 8
and 10) was selected on the basis of hypothesized interesting
polar interactions of the terminal amino group with three
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Scheme
1.
Synthesis
of
compounds
11-13a.
Enzyme
K
m
(RNA)
k
cat
k
cat/K
m
-fold
reduction
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b
(
µM)
a
-
(µM-1,
(
FU
·
i.
1
NH
N
min ) FU
·
O
min-1)
wild type
E256A
0.5 ± 0.1 1146
22
2278
1
±
14
15a-c
0.6± 0.2 126 ± 210
10.8
4.4
7.4
ii.
1
0
R262A
9 ± 2
4794
± 62
522
307
0
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7
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9
0
OH
OH
N
iii.
N
E256A/R262A 0.6
0.25
±
148±
70
NH3
NH2
CH3COO
1-13
m
Kinetic parameters K and kcat were calculated as described in
Materials and Methods, values represent the mean of three
independent experiments ± S.D.; a. FU, arbitrary fluorescence
m m
emission units; b. ratio kcat/K (wt)/kcat/K (mut).
1
5, 16a-b
a
Reagents and conditions. i. NaNH
ii. NH (g), DCM, 72 hours, from -78°C to r.t.; iii. CH
6 hours, r.t
2
, benzene, 12 hours, reflux;
3
3
COOH, DCM,
The E256A substitution had the greatest impact on the
enzymatic efficiency, causing a 10.8-fold reduction of the
k
cat/K
m
value for RNA unwinding, with respect to wild type
DDX3X. The R262A mutation also reduced the kcat/K value by
m
All three compounds showed greatly improved potencies with
respect to compound 5, (Figure S3) being able to suppress
DDX3X helicase activity in the low nanomolar range, likely due
to improved solubility (Table 1). These compounds were used
for all the subsequent experiments, as described below.
The modeling study predicted that compound 5 could make
polar interactions with the amino acids Glu256, Cys317 and
Tyr266, and hydrophobic interactions with Arg262, His318
and Pro247. In particular, the docking analysis allowed us to
hypothesize that the Glu256 and Arg262 residues, located at
the N- and C-terminal sides of the UM sequence, respectively,
as the major responsible for compound 5 binding. Being all
these structural considerations based on theoretical
assumptions stemming from our homology modelling, it was
necessary to experimentally validate them. Thus, the
compounds listed above were selected for further
investigation.
4.4- fold with respect to the wild type enzyme. Combining the
two mutations did not result in an additive effect and the
double mutant showed an intermediate phenotype (7.4-fold
m
reduction in the kcat/K value with respect to the wild type),
suggesting that the effect of the E256A mutation was not
changed by the presence of the additional R262A substitution.
The single mutants were also compared for their ATPase
activities. As shown in Fig S4, the E256A mutant showed
comparable activity with respect to the wild type enzyme,
while the ATPase activity of the R262A mutant was reduced,
similarly to the double E256A/R262A mutant. In summary,
these results indicated that the residue Glu256 is essential for
the RNA helicase, but not for the ATPase activity of DDX3X,
while Arg262 plays an ancillary role likely coupling ATP
hydrolysis to RNA unwinding. These results agree with
previously published data indicating a role of the DDX3X UM in
substrate binding and highlighting the central role of the
1
1
residue Glu256 in this motif for RNA interaction .
Glu 256 is essential for the RNA helicase activity of DDX3X
In an attempt to obtain experimental evidence of the reliability
of the molecular modeling predictions, the aminoacidic
residues Glu256 and Arg262 of DDX3X were changed into Ala
by site-directed mutagenesis to generate, respectively, the two
single E256A and R262A mutants and the double
E256A/R262A mutant (Figure S2). Next, to evaluate the impact
of these mutations on the enzymatic activity, the mutated
enzymes were tested for their RNA helicase activities in a
FRET-based assay and the kinetic parameters for the reaction
are reported in Table 2.
Glu 256 is the major determinant for the interaction of the UM
compounds with the DDX3X unique motif.
Next, the compounds 11 and 13, designed as described above
and hypothesized based on our theoretical in silico docking
studies to interact with the mutated residues, were tested for
their ability to inhibit the RNA helicase activity of DDX3X wild
type and mutant proteins. Even if the racemic compound 11
and its two enantiomers (compounds 12 and 13) showed
comparable activities, as reported in Table 1, we chose to test
1
1 and one of the two enantiomers to further confirm our data.
As shown in Table 3, the E256A substitution caused a reduction
of the inhibitory potencies of the compounds 11 and 13 of
36,500- and 43- fold, respectively. On the other hand, the
mutation R262A did not significantly change the inhibitory
potencies of the compounds, with respect to the wild type
enzyme. Thus, the residue Glu256 appeared as a major
determinant for inhibitor binding.
Table 2. Kinetic parameters for the RNA helicase
activity of DDX3X wild type and the DDX3X(E256A),
DDX3X(R262A) and DDX3X(E256A/R262A) mutants.
5
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Table 3 Inhibition of the RNA helicase activity of DDX3X
Page 6 of 13
13
58± 4.5
8,285)
>100
>100
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
wild type and the DDX3X(E256A) and DDX3X(R262A)
mutants.
(
(>10,000)
(>10,000)
Compound wild type
ID50, µMa
E256A
R262A
a. ID50, 50% inhibitory concentration. Values were calculated as
described in Materials and Methods and represent the mean of
three independent experiments ± S.D; b. ratio ID50/ID50(wt
DDX3X), the ID50 (wt DDX3X) values used were those reported in
Table 1.
ID50, µM
ID50, µM
(-fold
(-fold
resistance)
resistance)b
Finally, compounds 11, 12 and 13 were tested for their ability
to reduce WNV and DENV replication on Huh-7 cells. As
reported in Table 5, compound 11 and his enantiomers 12 and
1
1
0.002
.0005
±
±
73± 10
0.001± 0.0003
(0.5)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
(
36,500)
1
3, showed promising antiviral activities in the micromolar
range, resulting in IC50 values comparable (DENV) or even
significantly lower (WNV) than those shown by the broad-
spectrum antiviral ribavirin, without significant cytotoxicity
13
0.007
0.0001
0.3± 0.1
43)
0.004±0.002
(0.6)
(
(
see also Figure S5 and S6).
a. ID50, concentration of the compounds giving 50% inhibition of
the activity measured for the respective enzyme in the absence of
inhibitor. Values were calculated as described in Materials and
Methods and represent the mean of three independent
experiments ± S.D; b. ratio ID50(mut)/ID50(wt).
Table 5: Antiviral activities and cytotoxicity of selected
a
compounds against DENV and WNV .
Cpd ID
IC50b
IC50b
CC50c,d
(µM)
CC50c,e
(µM)
(DENV)
µM)
(WNV)
(µM)
These results were in good agreement with the molecular
modeling and the kinetic analysis (Table 2), which indicated
that Glu256 provides essential interactions of DDX3X with its
RNA substrate. The binding of the inhibitor to this key residue,
can be hypothesized to disrupt those interactions, providing a
possible structural basis for the selectivity of the mechanism of
action of our compounds.
(
#11
7.9±3.3
12.6±5.4
6.3±5.8
4.0±0.6
2.3±0.6
100±11.3
120±18.0
79±11.8
>100
>100
>100
>100
>100
#12
0.90±0.20
0.88±0.10
91.5±5
#13
ribavirin
a. Data represent mean + standard deviation of three
experiments; b. IC50: half maximal inhibitory concentration; c.
CC50: half maximal cytotoxic concentration; d. calculated using
CellTiter-Glo® kit; e. calculated using MTT kit; ribavirin was
used as a reference compound.
Selectivity and antiviral activity of UM compounds
To further prove the specificity of inhibition of the selected
compounds towards DDX3X, inhibition assays were performed
with three RNA helicases: the DEAD-box RNA helicase family
members human DDX1 and plant STRS2 from Arabidopsis
thaliana, and the DExH-family viral NS3 RNA helicase of HCV.
As shown in Table 4, all the compounds tested inhibited DDX1
with potencies almost four orders of magnitude lower than
towards DDX3X (selectivity ≈ 9,000- fold) and were completely
inactive against STRS2 and NS3, confirming the high selectivity
of inhibition for DDX3X. Again, these results provide support to
our theoretical framework, indicating that the desired
selectivity was apparently achieved.
Discussion and Conclusions
In this work, we attempted to develop a theoretical framework,
based on a homology model of the human DDX3X in its closed
conformation, to explore the possibility to develop molecules
targeting a unique structural domain of the protein. The main
aim of our work was to provide proof-of-principle for a strategy
potentially leading to the development of small molecules able
to couple broad-spectrum antiviral activity with absolute
selectivity for their molecular target. Such a seemingly
paradoxical goal (broad-spectrum activity vs. high selectivity
for the target), in fact, could be in principle achieved by
exploiting a specific cellular target, in our case the RNA helicase
DDX3X, identifying a unique structural domain present only in
the target, the UM domain of this study, and implementing
molecular modeling, in silico drug design and medicinal
chemistry techniques, to design compounds specifically
tailored on such a unique structural domain. An obvious
limitation of our work is the fact that no high-resolution
structures for the UM are presently available. We tried to
overcome such limitation by developing a homology model
using the best templates currently available. The coherence of
our homology-built model with the most recent crystal
structure of DDX3X in its active conformation was very high,
Table 4 Inhibition of the RNA helicase activity of human
DDX1, Arabidopsis STRS2 and HCV NS3.
Compound DDX1
ID50, µMa
STRS2
NS3
ID50, µM
ID50, µM
(-fold
selectivity)b
(-fold
selectivity)
(-fold
selectivity)
1
1
18±1.202
>100
>100
(
9,000)
(>50,000)
(>50,000)
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comforting us on the reliability of our approach. Our
number of infected people by the aforementioned viruses is
1
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hypothesized mode of binding was tested by in vitro
mutagenesis and biochemical assays, whose results further
corroborated the theoretical model here proposed, by
confirming the selectivity of the designed molecules. Finally,
cellular assays confirmed the antiviral activity as predicted.
A limited number of broad-spectrum antivirals targeting
different families of viruses (RNA viruses, DNA viruses and
retroviruses), have been reported in the scientific literature up
indeed raising. For example, the worldwide incidence of DENV
has risen 30-fold in the past 30 years, and more countries are
reporting their first outbreaks of the disease (source: WHO).
Consequently, it is important to develop new molecules acting
against these pathogens for which no specific drugs are
available up to date.
Classically, strategies to control these arboviral infections aim
at reducing the susceptibility to infection of the vector
(reduced competence/transmission) or the host (antiviral
drugs/vaccines). In this latter case, it would be greatly
desirable to identify a unique target whose inhibition could
counteract the infection of a wide range of arboviruses. Under
this respect, the cellular target DDX3X, central to the
replication of different viruses, will offer the possibility to
develop broad-spectrum antivirals, which are currently lacking
2
2-24
to now and only very few targeting cellular proteins
.
However, none of these compounds have been rationally
designed against its target, with the specific aim of achieving
optimal selectivity. Indeed, the main drawback of targeting a
cellular protein is the possibility of unwanted side effects,
either through the inhibition of the protein itself or by off-
targeting of the drug towards other cellular enzymes. This
problem is well known in the field of anticancer chemotherapy.
For example, Imatinib (also known as STI571 or Gleevec), is
one of the most successful molecular targeted chemotherapics
currently used in cancer therapy. This molecule was rationally
designed to target the ATP binding site of the tyrosine kinase
ABL, but it was subsequently shown to interact also with other
targets, most notably the receptor tyrosine kinases KIT and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
28
in our antiviral arsenal . The relevance and potential impact of
the approach disclosed here is further enhanced by the fact
that, besides being active against different viruses, an antiviral
agent selectively targeting a unique host factor could have the
additional advantage of overcoming the problem of drug
resistance. In fact, a cell harboring a mutated version of the
target protein will be more easily infected and thus either killed
by the virus or by the host immune system, without gaining a
fitness advantage. This, coupled with the low mutation rate of
cellular proteins, could result in a very high genetic barrier
towards drug resistance.
In conclusion, our results allow to hypothesize that the unique
domain of DDX3X could be used as a target for broad-spectrum
antiviral drugs endowed with maximal selectivity. This study
then paves the way for the development of larger focused
libraries targeting such domain, in order to expand SAR studies
and fully characterize their mode of interaction.
2
5
PDGFR (platelet-derived growth factor receptor) , thus failing
to achieve the desired selectivity.
The cellular ATPase/RNA helicase DDX3X is considered an
attractive molecular target for chemotherapy, since several
studies indicated that inhibiting DDX3X can have a therapeutic
efficacy both against cancer and viral infections, without
significant toxicity. In fact, as previously demonstrated by us,
1
5,
DDX3X inhibitors were very well tolerated in rats and mice
.
2
6
This is likely because DDX3X is part of a family of more than
5
0 related RNA helicases, which can have overlapping
16
compensatory roles in the cell metabolism . DDX3X is
required for the replication of several viruses, including HIV-1,
HCV, JEV, DENV and WNV
2
7-29
and in the last years, several
Experimental section
Homology Model
molecules were developed by our group against DDX3X,
providing proof-of-principle for its exploitation as a valuable
The homology model was obtained with the software PRIME
1
5
new target for broad-spectrum antiviral chemotherapy .
However, the significant similarity both from a sequence and a
structural point of view, in the ATP- and RNA-binding sites
among the members of the DEAD-box family of RNA
31
(version 3.7) , using the crystal structure of Drosophila Vasa
32
DEAD-box helicase (PDB ID: 2DB3) as the first template and
33
the open conformation of human DDX3X (PDB ID: 2I4I) as the
second template. This second template has been used to fill the
sequence of ten residues unique for the human DDX3 protein.
All other parameters of PRIME tools are setting as default
values. The obtained model was energy minimized by the
1
6
helicases , makes it difficult to achieve absolute selectivity for
DDX3X only by conventional drug design, leaving the problem
of possible off-targeting effects still open.
The UM series of compounds here described, represent the first
class of inhibitors of the human helicase DDX3X, rationally
designed to specifically recognize a unique motif (sequence
34
software MacroModel (version 10.5) , and it was also
validated using the Ramachandran plot.
ALRAMKENGRYGRRK- aa 250-264) of
a unique target
Docking procedure
(
DDX3X). This unique motif is not shared among other DExD-
The Asinex Gold and Platinum databases were screened using
box proteins, nor with other human proteins. These molecules
also showed no toxicity in cells and a considerable antiviral
effect being able to suppress the replication of WNV and DENV-
35
GLIDE software (version 6.4) with the HTVS procedure and
36
GOLD docking tools program (version 3.2) . The genetic
algorithm (GA) of GOLD allows for a partial protein Flexibility.
The box was centered on the Arg252 and has a radius of 9 Å.
The ChemScore was chosen as the fitness function. The search
efficiency was set to 100%. 100 independent GA runs were
performed, with a maximum number of 100000 GA operations
on a set of 5 islands with a population size of 200 individuals.
The remaining GA parameters were kept to their default values.
Pictures of the modeled ligand-enzyme complexes, together
with graphic manipulations, were rendered with the PyMOL
2
viruses in infected cells.
ZIKV, DENV, CHIKV, and other viruses are transmitted to
humans by the Aedes aegypti and Aedes albopictus mosquitoes.
More than half of the world’s population lives in areas where
these mosquito species are present and climatic and human-
caused changes can also expand the normal living niche of
these animals . The spread of the arthropod vectors and the
increased movement of people among different geographical
areas increase the risk of sudden outbreaks or even large
epidemics caused by these mosquito-borne viruses and the
3
0
37
package59 (version 1.5 [http://www.pymol.org/]) .
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Page 8 of 13
1
3
Molecular graphics and analyses were performed with UCSF
Chimera, developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California,
San Francisco, with support from NIH P41-GM103311 (version
3
2.61-2.52 (m, 1H). C NMR (101 MHz, CDCl ) δ 149.34 (2C),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
129.38(4C), 122.73 (2C), 121.04(4C), 53.02, 50.66, 46.18 ppm.
MS: (ESI) m/z 226.0 (M+H) .
+
3
8
1
.14 [http://www.rbvi.ucsf.edu/chimera]) .
General procedure for the synthesis of compounds 5, 16a-c
Gaseous ammonia was bubbled into a solution of the
opportune epoxide (0.1775 mmol) in DCM, in a sealed tube.
The corresponding mixture was stirred at room temperature
for 72h, then, volatiles were removed at reduced pressure, and
the crude was purified by flash chromatography on silica gel
(Purification Eluent: DCM/MeOH 9:1).
Methods. Reagents were obtained from commercial suppliers
Sigma-Aldrich and Alfa Aesar). All commercially available
chemicals were used as purchased without further purification.
CH Cl and Benzene were dried before use by distilling from
calcium hydride or sodium/benzophenone. Anhydrous
reactions were run under a positive pressure of dry N or
argon. Thin Layer Chromatography (TLC) was carried out using
Merck TLC plates silica gel 60 F254. Chromatographic
purifications were performed on columns packed with Merk 60
silica gel, 23-400 mesh, for flash technique.
(
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
1-amino-3-(diphenylamino)propan-2-ol (16a), H NMR
3
(400 MHz, CDCl ) δ 7.22 (t, J = 7.6 Hz, 4H), 7.00 (d, J = 7.8 Hz,
4H), 6.93 (t, J = 7.1 Hz, 2H), 3.88 (m, 1H), 3.70 (d, J = 6.0 Hz, 2H),
1
3
2.94 (m, 3H), 2.83 (d, J = 12.3 Hz, 1H), 2.67 – 2.53 (m, 1H).
NMR (101 MHz, CDCl ) δ 148.35 (2C), 129.40 (4C), 121.81 (2C),
121.30 (4C), 68.86, 56.45, 44.57.
(S)-1-amino-3-(diphenylamino)propan-2-ol (16b), H NMR
(400 MHz, CDCl ) δ 7.22 (t, J = 7.6 Hz, 4H), 7.00 (d, J = 7.8 Hz,
4H), 6.93 (t, J = 7.1 Hz, 2H), 3.88 (m, 1H), 3.70 (d, J = 6.0 Hz, 2H),
C
3
Instrumentation. All NMR spectra were recorded on Bruker
1
1
Avance DPX400 spectrometer at 400 MHz for H-NMR or 100
13
MHz for C-NMR. Chemical shifts are reported relative to
3
tetramethylsilane at 0.00 ppm. Mass spectra (MS) data were
obtained using an Agilent 1100 LC/MSD VL system (G1946C)
with a 0.4 mL/min flow rate using a binary solvent system 25
of 95:5 methyl alcohol/ water. UV detection was monitored at
1
3
2.94 (m, 3H), 2.83 (d, J = 12.3 Hz, 1H), 2.67 – 2.53 (m, 1H).
NMR (101 MHz, CDCl ) δ 148.35 (2C), 129.40 (4C), 121.81 (2C),
121.30 (4C), 68.86, 56.45, 44.57.
(R)-1-amino-3-(diphenylamino)propan-2-ol (16c), H NMR
(400 MHz, CDCl ) δ 7.22 (t, J = 7.6 Hz, 4H), 7.00 (d, J = 7.8 Hz,
4H), 6.93 (t, J = 7.1 Hz, 2H), 3.88 (m, 1H), 3.70 (d, J = 6.0 Hz, 2H),
C
3
1
2
54 nm. Mass spectra were acquired in positive and negative
mode scanning over the mass range. Microwave irradiation
experiments were conducted using CEM Discover Synthesis
Unit (CEM Corp., Matthews, NC). For the quantitative analysis
was used a UV/LC-MS system. LC analysis was performed by
Agilent 1100 LC/MSD VL system (G1946C) (Agilent
Technologies, Palo Alto, CA). Spectra were acquired over the
scan range m/z 50-1500 using a step size of 0.1 u. The purity of
compounds (as measured by peak area ratio) was >97%.
3
13
2.94 (m, 3H), 2.83 (d, J = 12.3 Hz, 1H), 2.67 – 2.53 (m, 1H).
C
NMR (101 MHz, CDCl ) δ 148.35 (2C), 129.40 (4C), 121.81 (2C),
3
121.30 (4C), 68.86, 56.45, 44.57.
General procedure for the synthesis of compounds 11-13
The reaction is summarized in Scheme 1. Compounds 16a-c
were solubilized in 3 mL of dichloromethane, 500 µL of acetic
acid were added, and the resulting mixture was stirred at room
temperature for 6 hours. The solvent was removed in vacuum
e the resulting mixture crystallized from acetonitrile.
Chemistry
General procedure for the synthesis of compounds 15a-c
Diphenylamine (1.1818 mmol) was added to a suspension of
2
NaNH (3.54 mmol) in anhydrous benzene. After 30 min
1-amino-3-(diphenylamino)propan-2-ol acetate (11),
1
epichlorohydrin or (R)-(−)-epichlorohydrin or (S)-(+)-
epichlorohydrin (1.18 mmol) was added, and the reaction was
stirred at reflux for 12 hours. After this time pH was adjusted
to 7 with HCl 1N and the mixture extracted with EtOAc
Aspect white solid, Y= 85% H NMR (400 MHz, D
2
O) δ 7.29-
7.25 (m, 4H), 7.01-6.96 (m, 6H), 4.16-4.01 (m, 1H), 3.82-3.71
1
3
(m, 2H), 3.15-3.11 (m, 1H), 2.892.83 (m, 1H), 1.84 (s, 3H).
C
NMR (101 MHz, D O) δ 171.16, 148.35 (2C), 129.40 (4C),
2
(
3x25mL). The organic layers were collected, washed with
Brine and dried over Na SO . The crude was purified by flash
chromatography on silica gel using the opportune eluent.
121.81 (2C), 121.30 (4C), 68.86, 56.45, 44.57, 23.12 ppm.
(S)-1-amino-3-(diphenylamino)propan-2-ol acetate (12),
2
4
1
Aspect white solid, Y= 90%, H NMR (400 MHz, D
2
O) δ 7.29-
N-(oxiran-2-ylmethyl)-N-phenylaniline(15a),
colourless oil, Y= 89%. Eluent PE/EA=8/2 H NMR (400 MHz,
Aspect
7.25 (m, 4H), 7.01-6.96 (m, 6H), 4.16-4.01 (m, 1H), 3.82-3.71
1
13
(m, 2H), 3.15-3.11 (m, 1H), 2.892.83 (m, 1H), 1.84 (s, 3H).
NMR (101 MHz, D O) δ 171.16, 148.35 (2C), 129.40 (4C),
121.81 (2C), 121.30 (4C), 68.86, 56.45, 44.57, 23.12 ppm.
(R)-1-amino-3-(diphenylamino)propan-2-ol acetate (13),
C
3
CDCl ) δ 7.26 (d, J = 8.1 Hz, 4H), 7.05 (d, J = 7.6 Hz, 4H), 6.96 (t,
2
J = 7.8 Hz, 2H), 4.12 – 3.77 (m, 2H), 3.30 – 3.11 (m, 1H), 2.81-
1
3
2
1
5
.70 (m 1H), 2.61-2.52 (m, 1H). C NMR (101 MHz, CDCl
3
) δ
1
49.34 (2C), 129.38(4C), 122.73 (2C), 121.04(4C), 53.02,
0.66, 46.18 ppm. MS: (ESI) m/z 226.0 (M+H) .
Aspect white solid, Y= 87%, H NMR (400 MHz, D
2
O) δ 7.29-
+
7.25 (m, 4H), 7.01-6.96 (m, 6H), 4.16-4.01 (m, 1H), 3.82-3.71
1
3
(
S)-N-(oxiran-2-ylmethyl)-N-phenylaniline(15b), Aspect
(m, 2H), 3.15-3.11 (m, 1H), 2.892.83 (m, 1H), 1.84 (s, 3H).
C
1
colourless oil, Y= 85% PE/EA=8/2 H NMR (400 MHz, CDCl
3
) δ
NMR (101 MHz, D O) δ 171.16, 148.35 (2C), 129.40 (4C),
2
7.26 (d, J = 8.1 Hz, 4H), 7.05 (d, J = 7.6 Hz, 4H), 6.96 (t, J = 7.8 Hz,
121.81 (2C), 121.30 (4C),68.86, 56.45, 44.57, 23.12 ppm.
2
2
1
H), 4.12 – 3.77 (m, 2H), 3.30 – 3.11 (m, 1H), 2.81-2.70 (m 1H),
1
3
.61-2.52 (m, 1H). C NMR (101 MHz, CDCl
3
) δ 149.34 (2C),
Site-directed mutagenesis
29.38(4C), 122.73 (2C), 121.04(4C), 53.02, 50.66, 46.18 ppm.
The double mutant E256A/R262A was obtained by Biofab
Research SRL (Rome, Italy) and cloned into a pET30a
(Novagen) expression vector. The two single mutants E256A
and R262A were obtained by site-directed mutagenesis as
+
MS: (ESI) m/z 226.0 (M+H) .
R)-N-(oxiran-2-ylmethyl)-N-phenylaniline(15c), Aspect
(
1
3
colourless oil, Y= 82% PE/EA=8/2 H NMR (400 MHz, CDCl ) δ
3
9
7.26 (d, J = 8.1 Hz, 4H), 7.05 (d, J = 7.6 Hz, 4H), 6.96 (t, J = 7.8 Hz,
2H), 4.12 – 3.77 (m, 2H), 3.30 – 3.11 (m, 1H), 2.81-2.70 (m 1H),
described .
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Specifically, the DDX3X double mutant E256A/R262A cDNA
The double mutant protein E256A/R262A and the single
1
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6
7
8
9
1
1
1
1
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1
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1
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2
2
2
2
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5
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6
cloned into pET30a was used to obtain the single mutants.
Couples of primers of 36/42 bp overlapping for the whole
length except for a nucleotide, were used that reverts one of the
two alanines into the previous wild type amino acid. To quickly
verify the introduction of the correct mutation, a restriction
site of SfiI was removed from the gene sequence of single
mutants, without introducing any additional amino acid
substitution.
mutants E256A and R262A were cloned in the E. coli
expression vector pET-30a(+) and BL21(DE3) cells were
transformed with the plasmids. Proteins induction and
purification were the same as the full-length protein. Only the
double mutant E256A/R262A was dialysed as the wild-type
enzyme, whereas the single mutants E256A and R262A were
maintained in elution buffer avoiding further loss of material
due to the dialysis procedure.
The sequences of the primers were as follows (primer-primer
overlapping sequences are underlined):
Helicase assay based on Fluorescence Resonance Energy
Transfer (FRET)
E256A fw 5’ tatgggcgccgcaaacaatacccaatctccttggtattagca3’
E256A rv 5’ gcggcgcccataccttccattggccttcattgccct3’
R262Afw 5’ atgaaggaaaatggaaggtatggggcccgcaaacaa3’
R262A rv 5’ attttccttcattgccctcaaagcctcgcctggacc3’
The mutagenesis was carried out in the following reaction
mixture: Pfu buffer 1X (Promega), dNTPs 0.2 mM, Primers 1 µM
and 3 U of Pfu (Promega).
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The FRET helicase assay was performed as described . Briefly,
the dsRNA substrate for the helicase assay was prepared by
hybridizing two ss RNA oligonucleotides with the following
sequences:
Fluo-FAM
UUUUUUUUUUUUUUAGUACCGCCACCCUCAGAACC 3’
Qu-BHQ1 5’ GGUUCUGAGGGUGGCGGUACUA 3’
5’
The PCR cycles were: 94°C for 2 minutes (to denature the
template DNA); 3 amplification cycles at 94°C for 1 minute,
DNA capture 5’ TAGTACCGCCACCCTCAGAACC 3’
The sequence in Fluo-FAM complementary to Qu-BHQ1 is
6
1
2°C for 1 minute and 68°C for 12 minutes. After that additional
2 amplification cycles were performed as follows: 94°C for 1
underlined. Flu-FAM carries
a
6- carboxyfluorescein
minute, 57°C for 1 minute and 68°C for 12 minutes. The PCR
cycles were followed by an extension step at 68°C for 60
minutes.
fluorophore at its 3' end, while Qu-BHQ1 carries a Black Hole
quencher group at its 5' end. The DNA capture oligonucleotide
is complementary to the Qu-BHQ1 oligonucleotide but bears no
modifications.
Helicase assay using the dsRNA substrate was performed in 20
2
mM TrisHCl (pH 8), 70 mM KCl, 2 mM MgCl , 2 mM
The PCR products were treated with 10 units of DpnI
(Promega) at 37°C for 2 hours and then 10 µl of each PCR
reaction were analyzed by agarose gel electrophoresis.
Amplified DNA was transformed into E. coli DH5α competent
cells by heat shock following standard protocol. The
transformed cells were spread on a LB plate containing 50
µg/µl Kanamycin and incubated at 37°C overnight. Colonies
from each plate were grown and the plasmid DNA was isolated.
To verify the mutations, 400 ng of plasmid DNA was first
linearized with 10 units of Hind III (Promega) for 1 hour at 37
°C and then digested with 12 units of Sfi I (New England
Biolabs) for 5 hours at 50 °C. Positive colonies were sent to
Eurofins Genomics (Ebersberg, Germany) for Sanger
sequencing to verify correct mutations insertions.
dithiothreititol, 12 units RNasin (Promega), 2 mM ATP, 50 nM
dsRNA and 100 nM capture strand in 20 µl of reaction volume.
The unwinding reaction was started by adding 60 pmols of
DDX3X recombinant protein and carried out at 37°C for 40 min
using a LightCycler 480 (Roche). The fluorescence intensity
was recorded every 30 s. Data of fluorescence signals were
analysed by linear interpolation and the corresponding slope
values were used to determine the apparent unwinding rate.
The same assay was used to test all the inhibitor molecules
against DDX3X helicase activity. In the negative controls, we
replaced the enzyme with dialysis buffer and the inhibitor with
DMSO. In the positive control, the inhibitor is replaced with
DMSO to detect the unwinding activity of DDX3X enzyme only.
This latter activity was taken as 100% enzymatic activity and
compare to enzymatic activities in the presence of different
inhibitor concentrations to calculate ID50 values.
Proteins production and purification
Purification of human recombinant full-length DDX3X was as
2
6
described . Briefly, DDX3X cDNA was cloned in the E. coli
expression vector pET-30a(+). ShuffleT7 E. coli cells were
transformed with the plasmid and grown at 37°C up to OD600
=
0
.7. DDX3X expression was induced with 0.5 mM IPTG at 15°C
ATPase assay
1
1
O/N. Cells were harvested by centrifugation, lysed and the
crude extract centrifuged at 100.000xg for 60 min at 4°C in a
Beckman centrifuge before being loaded onto a FPLC Ni-NTA
column (GE Healthcare). Column was equilibrated in Buffer A
(50 mM Tris-HCl pH 8.0, 250 mM NaCl, 25 mM Imidazole and
20% glycerol). After extensive washing in Buffer A, the column
was eluted with a linear gradient in Buffer A from 25 mM to 250
mM Imidazole. Proteins in the eluted fractions were visualized
on SDS-PAGE and tested for the presence of DDX3X by Western
blot with anti-DDX3X A300-474A (Bethyl Laboratories) at
The ATPase activity was determined, as previously described ,
by directly monitoring (γ-32P) ATP hydrolysis by thin-layer
chromatography (TLC). (γ-32P) ATP (3000Ci/mmol) and TLC
PEI Cellulose F plates were purchased from Hartmann Analytic
and Merck respectively.
The reaction was carried out in a final volume of 5 µl which
contained: different amount of DDX3X proteins as specified in
the Figures Legends, 0.1 µM (γ-32P) ATP (3000 Ci/mmol) as
2
tracer plus 1 µM of cold ATP, 5 mM MgCl and nucleic acids as
indicated. Samples were incubated for 30 minutes at room
temperature and 1.5 µl of the reaction were dotted onto TLC
sheets of polyethylenimine cellulose. The products were
1
:4000 dilution in 5% milk. Fractions containing the purest
DDX3X protein were pooled and dialyzed in Slide-A-Lyzer®
MINI Dialysis Devices 20K MWCO (Thermo Scientific) for 3
hours (25 mM Tris HCl pH 8.0, 0.5 mM DTT, 100 mM NaCl,
Glycerol 20%) in order to remove the salt excess. Finally,
proteins were stored at -80°C.
2 4
separated by ascending chromatography with 0.5 M KH PO
(pH 3.4). The intensities of the radioactive bands
corresponding to ATP and Pi were quantified by densitometric
scanning with PhosphoImager (Typhoon, Ge Healthcare).
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The ATPase activity was also determined using the commercial
kit ADP-Glo Kinase Assay (Promega) as previously described
when the cell layers were confluent, the medium was removed,
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the wells were washed twice with PBS, treated with 100 ml of
DMEM with 10 ml DMSO alone (cell positive control) or with
serial tenfold dilutions of DDX3X inhibitors (final
concentrations of 100, 10, 1, 0.1, and 0.01 µM) and incubated
1
5
in . Reaction was performed in 30 mM TrisHCl pH 8, 9 mM
MgCl , 0.05 mg/mL BSA, 50 µM ATP, nucleic acids as indicated
2
and different amounts of DDX3X proteins specified in the
figures. The reaction was performed following the ADP-Glo
Kinase Assay Protocol and luminescence was measured with
MicroBetaTriLux (Perkin-Elmer).
2
for 3 days at 37°C in a CO . After treatment, an MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) kit
(Roche, Milan, Italy) was used according to the supplier’s
instructions, and the absorbance of each well was determined
using a microplate spectrophotometer at a wavelength of 570
nm. Cytotoxicity was calculated by dividing the average optical
density of treated samples by the average of DMSO-treated
samples (cell positive control).
Kinetic analysis
Data (in triplicate) were plotted and analyzed by least-squares
nonlinear regression, with the computer program GraphPad
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Prism 6.0. Data were fitted to the following equation:
n
(1)
v=Vmax/(1+(K
m
/[S] ))
Where v is the apparent reaction velocity at each substrate
concentration, Vmax is the maximum rate of the reaction, K is
Cytotoxicity assays-CellTiter-Glo
Ribavirin
(1-β-D-Ribofuranosyl-1,2,4-Triazole-3-
m
the Michaelis-Menten constant, S is the variable substrate
concentration, n is an exponential term to take into account
sigmoidal dose–response curves, due to the positive
Carboxamide; Sigma Aldrich cat. R9644), used as a reference
compound, was supplied as a powder and dissolved in 100%
dimethyl sulfoxide (DMSO).
4
0
cooperativity of DDX3X binding to RNA .
Cytotoxicity of investigational compounds was determined by
incubating 7,000 Huh7 cells/well in presence of serial two-fold
dilution of compounds. Similarly, dilutions of DMSO were
added to cells to evaluate the cytotoxicity of the solvent used to
resuspend tested compounds. After 72 h incubation, cells were
treated with the CellTiter-Glo 2.0 Luminescent Cell Viability
Assay (Promega) according to the manufacturer’s protocol. The
luminescence values obtained from cells treated with
investigational compounds or DMSO were measured through
the GloMax® Discover Multimode Microplate Reader
(Promega) and elaborated with the GraphPad PRISM software
version 6.0 (La Jolla, California, USA) to calculate the half-
maximal cytotoxic concentration (CC50).
The turnover constant kcat was calculated as Vmax/(enzyme
concentration) and the RNA substrate binding efficiency of our
enzymes was calculated as the kcat/K ratio.
m
For the inhibition assays, the IC50 values have been calculated
from dose–response curves. Data (in triplicate) were plotted
and analyzed by least-squares nonlinear regression, according
to the method of Marquardt–Levenberg, with the computer
program GraphPad Prism 6.0. Data were fitted to the following
equation:
obs = Emax/(1+ (IC50/[I])n)
(2)
E
where E(obs) is the observed enzymatic activity in the presence
of each inhibitor dose [I]; E(max) is the maximal enzymatic
activity in the absence of the inhibitor; n is an exponential term
to take into account sigmoidal dose–response curves.
WNV inhibitory viral plaque reduction assay
Huh7 cells were used for the inhibitory viral plaque reduction
assay. WNV was used to infect Huh7 cell line in duplicate and
viral plaques were visualized 4 days following infection.
Cell lines and viruses
The human hepatoma cell line Huh7 (kindly provided by
Istituto Toscano Tumori, Core Research Laboratory, Siena,
Italy) and the African green monkey kidney cell line Vero E6
5
Briefly, 6-well plates were seeded with 2.5 × 10 cells in 3 mL
2
of growth medium and kept overnight at 37°C with 5% CO . The
day of infection, after removal of growth medium, cell
monolayers at 80–90% confluence were infected with WNV
viral stock with a multiplicity of infection (MOI) of 0.1 in a final
(
ATCC® no. CRL-1586™) were cultured in high glucose
Dulbecco's Modified Eagle's Medium with sodium pyruvate and
L-glutamine (DMEM; Euroclone) supplemented with 10% Fetal
2
volume of 0.3 mL and incubated for 1 h at 37°C with 5% CO .
Bovine
Penicillin/Streptomycin (Pen/Strep, Euroclone) and incubated
at 37°C in a humidified incubator supplemented with 5% CO
The Aedes albopictus mosquito cell line C6/36 (ATCC® no. CRL-
660™) was cultured in DMEM as Huh7 and Vero E6 with
Serum
(FBS;
Euroclone)
and
1%
Then, cells were washed with PBS 1X, and 30 mL
dimethylsulfoxide (DMSO) alone (viral positive control) or
with 10-fold serial dilutions of DDX3X inhibitory compounds
were immediately added in duplicates together with 300 ml of
fresh DMEM complete medium (compound final
concentrations of 100, 10, 1, 0.1, and 0.01 µM). Ribavirin (1-β-
2
.
1
additional L-glutamine (Euroclone, final concentration 6 mM)
and HEPES (Euroclone, final concentration 25 mM) and
incubated at 28°C. The New Guinea C DENV serotype 2 strain
was used for all the experiments described in this work and
was kindly provided by the Istituto Superiore di Sanità, Rome,
Italy. DENV strain was propagated in C6/36 cells and titrated
by plaque assay in Vero E6. WNV (strain of lineage 2, kindly
provided by Istituto Superiore di Sanità, Rome, Italy) viral
stock, consisting of cell-free supernatants of acutely infected
Huh7 cells, was aliquoted and stored at -80°C until used.
Titration of the viral stocks as plaque-forming unit (PFU) was
carried out in Huh7 cells.
d-Ribofuranosyl-1,2,4-Triazole-3-Carboxamide;
SIGMA)
diluted in DMSO was used as an inhibitory reference control.
Then, the overlay medium composed by 0.5% Sea Plaque
Agarose (Lonza, Basel, Switzerland) diluted in propagation
medium was added to each well. After 4 days (Huh7) of
incubation at 37°C, the monolayers were fixed with methanol
(
Carlo Erba Chemicals, Milan, Italy) and stained with 0.1%
crystal violet (Carlo Erba Chemicals) and the viral titers were
calculated by Plaque forming unit (PFU) counting. Percent of
Plaque reduction activity was calculated by dividing the
average PFU of treated samples by the average of DMSO-
treated samples (viral positive control). Fifty percent
Cytotoxicity assay-MTT
Monolayers of 2.5 × 10 Huh7 cells per well in flat-bottom 96-
4
inhibitory concentrations (IC ) were calculated using the
50
predicted exponential growth function in Microsoft Excel,
well culture plates and allowed to adhere overnight. Then,
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Journal of Medicinal Chemistry
which uses existing x-y data to estimate the corresponding anti
DDX3X compound concentration (x) from a known value (y),
which in this case was 50% PFU. Mean IC50 ± standard
deviations (SD) were calculated using all replicates. All
experiments were repeated at least twice and all experimental
Corresponding Author
Corresponding author: Giovanni Maga
E-mail: giovanni.maga@igm.cnr.it, ORCID: 0000-00018092-
552
Present Addresses
Current address: CIBIO, University of Trento, Trento, Italy.
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*
1
procedures were conducted under biosafety level
containment.
3
†
Deceased Author
¶
DENV Immuno-detection assay
Deceased, August 2019.
Immunodetection assay (IA) was designed to determine the
antiviral activity of candidate antivirals on the whole viral
Author Contributions
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replication cycle . Briefly, 7,000 Huh7/well were infected with
0 TCID50 of DENV. Viral adsorption was performed in 96-well
plates for 1 h at 37°C with 5% CO and after virus removal,
serial dilutions of each compound tested were added to the
cells and incubated at 37°C with 5% CO . In each test, ribavirin
‡ These authors equally contributed to this work and should be
considered as joint first authors.
5
2
GM and MB had the original idea, secured funding and
supervised the work together with ED. VR and AG purified
DDX3X and planned and performed all the enzymatic assays.
FC contributed to the enzymatic assays. ABr, RF and CIT
designed compounds. ABr synthesized compounds. ABo, FS, IV
e and MZ planned and performed cell-based assays with DENV.
FM and SG planned and performed cell-based assays with WNV.
VR wrote the manuscript together with GM, with the help of
ABr and AG.
2
was used as control of the inhibition of viral replication and
infected and uninfected cells without drugs were used to
calculate the 100% and 0% of viral replication, respectively.
After 72 h, supernatants were harvested and triplicate
dilutions of generated viruses were used to infect pre-seeded
2
Huh7 cells (7,000 cells/well) for 72 h at 37°C with 5% CO .
The supernatants were removed and cells were fixed for 30
minutes with 10% formaldehyde (Carlo Erba), rinsed with 1%
PBS and permeabilized for 10 minutes with 1% Triton X-100
Funding Sources
GM and VR were partially supported by the CNR Project
"Identification of novel molecules with therapeutic potential
against Zika virus and other emerging viruses"
(
Carlo Erba). Cells were washed with PBS containing 0.05%
Tween 20 (Carlo Erba) and incubated for 1 h with monoclonal
anti-flavivirus mouse antibody (clone D1-4G2-4-15; Novus
Bio) diluted 1:400 in blocking buffer (PBS containing 1% BSA
and 0.1% Tween 20). After washing, cells were incubated for 1
h with a polyclonal Horseradish Peroxidase (HRP)-coupled
anti-mouse IgG secondary antibody (Novus Bio NB7570)
diluted 1:10.000 in blocking buffer. Next, cells were washed
and the 3,3’,5,5’-Tetramethylbenzidine substrate (TMB, Sigma
Aldrich) was added to each well. After 15 minutes of incubation
in the dark, the TMB peroxidase-based reaction was stopped
with one volume of 0.5 M sulfuric acid. All incubation steps
were performed at room temperature. Absorbance was
measured at 450nm optical density (OD450) using the
Absorbance Module of the GloMax® Discover Multimode
Microplate Reader (Promega).
Notes
The authors declare no competing financial interests. All
original raw data are available upon request by the authors.
Published materials are available upon request, an MTA is
required for distribution of the compounds here disclosed.
ACKNOWLEDGMENT
This work is dedicated to the memory of Prof. Maurizio Botta,
who was a founder and a leading mind of this project and
transmitted his devotion for science to generations of
researchers.
Each IA run was validated by the OD450 value above 1 in the
virus control culture. OD450 values from each well were
normalized according to the 100% and 0% of viral replication
and normalized values were used to calculate half-maximal
inhibitory concentration (IC50) values through a non-linear
regression analysis of the dose-response curves generated
with GraphPad PRISM software version 6.0.
ABBREVIATIONS
DENV, Dengue virus; WNV, West-Nile virus; JEV, Japanese
encephalitis virus; CHIKV, Chikungunya virus; ZIKV, Zika virus;
DDX3X, human X-linked DEAD-box protein 3; HIV-1, Human
immunodeficiency virus type 1; HCV, Hepatitis C virus; UM,
unique motif; DDX1, human DEAD-box protein 1; STRS2, stress
response suppressor 2; NS3, HCV non-structural protein 3;
PDGFR, platelet-derived growth factor receptor.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is
available free of charge on the ACS Publications website at DOI:
Molecular formula strings (CSV)
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